Project Summary/Abstract Our laboratory focuses on the biology and mechanisms of gene silencing by DNA methylation and histone modifications, primarily using Arabidopsis thaliana as a model system. Recent efforts have focused on the mechanisms by which DNA methylation is properly patterned in the genome. The main findings are that four distinct DNA methylation pathways, driven by four different DNA methyltransferases act in self- reinforcing mechanisms to maintain cytosine methylation in three different sequence contexts, CG, CHG and CHH (where H = A, T, or C). The establishment of DNA methylation is controlled by the RNA-directed DNA methylation pathway, and studying this pathway has been a major recent focus of the lab. Most recently, the laboratory has used genetics, genomics, proteomics, and structural biology approaches to characterize the mechanisms by which two RNA polymerases, Pol IV and Pol V, act to produce non- coding RNAs that act to target RNA-directed DNA methylation to specific loci. The newest findings from the laboratory are also taking us into new directions aimed at understanding factors that act downstream of DNA methylation, to both control gene expression and to maintain genome stability. In the next five years, we plan to utilize all available approaches to understand the proteins that recognize methylated DNA and certain modified histones, and to decipher their functions in gene expression and in the maintenance of genome stability. These approaches will include mass spectrometry to directly identify proteins and protein complexes able to bind to methylated DNA and methylated histones. The laboratory will also use Arabidopsis mutant screens to identify factors that act downstream of DNA methylation and various histone marks. A post translational modification of histones called H3K27 monomethylation is critical for repression of heterochromatin. In the absence of this mark, cells undergo both inappropriate gene expression and a massive DNA damage response resulting in amplification of heterochromatic DNA. A key part of the five-year plan will be to understand the mechanisms at play, and to understand the precise nature of the relationship between gene derepression and DNA damage.